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. 2015 Jul 29;35(30):10773–10785. doi: 10.1523/JNEUROSCI.0099-15.2015

Figure 5.

Figure 5.

In vivo modification of HDB cholinergic neuron activity affects natural odor discrimination. A, Top left, Schematic diagram for the virus injection and behavioral testing schedule. Bottom left, Confocal image of a sagittal section of the OB from a ChAT-Cre mouse expressing hM4Di (red, mCherry) in the HDB. Dotted box represents the region shown on the right pictures (1,2). A1, A2, Magnified HDB sections immunostained for ChAT (green) and mCherry (red) showing colocalization (yellow) with hM3Dq (1) and hM4Di (2). Scale bar, 25 μm. B, Top, Recording from an HDB neuron expressing the hM4Di DREADD in the presence of iGluR blockers (APV 100 μm, CNQX 10 μm) and GABAzine (5 μm). Application of CNO (5 μm) produced a hyperpolarization in this cell (Vm = −54 mV). Calibration: 20 mV, 1 min. Bottom left, HDB neurons expressing the hM3Dq DREADD, loaded with the calcium dye Fluo-4. Dotted lines outline the HDB. Colored circles represent selected cells within the HDB (yellow, green, blue, and purple) responding to CNO. Red circle represents a cell outside the HDB. Bottom right, Optical recording traces color-coded to the cells shown on the left; cells in the HDB show an increase in calcium signal in the presence of CNO (5 μm). Calibration: 10% ΔF/F0, 2 min. C, Left, Habituation/dishabituation protocol used to test natural discrimination of odors. Mice presented with the same odor (i.e., ethyl heptanoate, C7, pink) three times show a decrease in investigation time (habituation). On the fourth trial, a novel odor (i.e., ethyl octanoate, C8, red) is presented and investigation time increases (dishabituation). The dotted box (i) highlights the quantification of habituation/dishabituation for this odor set (C7/C8), which is used to determine the discrimination of odors pairs in the middle and right graphs. Middle, ChAT-hM4Di mice were tested for natural discrimination of the C7/C8 (pink/red) and C6/C8 (purple/red) odor pairs (ethyl hexanoate, C6, purple). Odor discrimination was assessed before CNO injection (Control, PBS injected), CNO injection (CNO), and 5 h after CNO (Wash). Right, ChAT-hM3Dq mice were similarly tested for olfactory discrimination with the C7/C8 odor pair and carvone isomers: dark blue represents l-carvone; light blue represents d-carvone. **p < 0.02. ***p < 0.01.